1. Field of the Invention
The present invention relates to an optical microscope for observing a sample by using nonlinear optical response process.
2. Description of the Related Art
The technique of optical microscopes has a long history, during which various types of optical microscopes have been developed. In recent years, as a result of progress in the peripheral technologies such as laser technology and electronic imaging technology, high-performance optical microscope systems have been developed.
In such a background, high-performance optical microscopes using various spectral processes have been proposed, so that not only a shape of a sample can be analyzed but also a molecule included in the sample can be identified and/or the structure thereof can be analyzed. In recent years, in particular, microscopy detecting response light specific to nonlinear optical response process has been attracting attention since it is capable of easily inducing a nonlinear optical response process with respect to a substance by using pulsed laser having high peak power. Examples of representative nonlinear optical response process include: 1. Raman process; 2. harmonics process (particularly, SHG process); and 3. multiphoton absorption process (particularly, two-photon absorption process). Hereinafter, the respective processes will be briefly explained.
1. Raman Processes
In Raman process, there is Raman spectroscopy in which photoresponse from a biological sample or an industrial material is observed without staining the biological sample or the industrial material to analyze the structure thereof. Raman spectroscopy is expected to be applied to the field of a microscope (see JP2002520612, for example). Raman spectroscopy is based on a type of nonlinear optical effect referred to as Raman effect. When incident light is scattered by a molecule or an atom at high photon flux, a quantum state of the molecule is changed and energy of the entire system is changed. At that time, the changed energy shifts to the scattered photon, whereby, light having a different wavelength from that of the incident light appears. Such a phenomenon is referred to as Raman scattering. As Raman scattering, there are known three types of Raman scattering with the use of monochromatic light, that is, (1) non-resonant Raman scattering, (2) true resonance Raman scattering, and (3) preresonance Raman scattering and one type of Raman scattering with the use of polychromatic light, that is, (4) coherent Raman scattering. Hereinafter, these types of Raman scattering will be further described with reference to FIGS. 24 and 25.
(1) Non-Resonant Raman Scattering
FIG. 24(a) is an energy diagram explaining non-resonant Raman scattering. Non-resonant Raman scattering can be explained by second-order perturbation theory from a viewpoint of an atom and a molecule. Specifically, as shown in FIG. 24(a), non-resonant Raman scattering corresponds to a kind of two-photon process in which a virtual quantum level of S (imaginary) is assumed. In this two-photon excitation process, molecules in a lowest electronic state and a lowest rovibrational level, that is, in the ground state (state S0) are once excited to a virtual quantum level S (imaginary) by, for example, extremely strong laser light and afterwards, de-excited to a high rovibrational level (V2) of the lowest electronic state. As a result, as is apparent from FIG. 24(a), the incident light provides an atom or a molecule with photon energy of (Ei-E0), whereby light after non-resonant Raman scattering loses photon energy accordingly and the light is scattered such that the initial wavelength λ1 thereof is apparently changed to a longer wavelength of λ2. In general, in a two-photon process based on a virtual quantum level including non-resonant Raman scattering, transition probability is extremely small. In order to induce this process, a super short pulsed laser in the order of femtosecond may be required.
(2) True Resonance Raman Scattering
FIG. 24(b) is an energy diagram explaining true resonance Raman scattering. True resonance Raman scattering is a scattering process in which non-resonance Raman scattering satisfies a specific condition that S (imaginary) coincides with the actual first electronically-excited state S1, as shown in FIG. 24(b). This case corresponds to a process in which a molecule of the ground state S0 is excited to the actual first electronically-excited state S1 and then de-excited to a high rovibrational level (V2) of a lowest electronic state. The process, as shown in FIG. 24(b), apparently corresponds to a process in which fluorescence of λ2 was emitted after S0→S1 excitation. Since this true resonance Raman scattering utilizes the actual quantum states scattering probability is extremely high and Raman scattering can be achieved with markedly high light intensity as compared with non-resonant Raman scattering.
(3) Preresonance Raman Scattering
FIG. 24(c) is an energy diagram explaining preresonance Raman scattering. Preresonance Raman scattering has a property intermediate between true resonance Raman scattering and non-resonant Raman scattering. Specifically, this is a case where a level of S (imaginary) exists in the vicinity of the first electronically-excited state S1.
(4) Coherent Raman Scattering
FIG. 25 is a view explaining coherent Raman scattering. FIG. 25(a) is an energy diagram of Coherent Anti-Stokes Raman Scattering (CARS) and FIG. 25(b) is an energy diagram of Coherent Stokes Raman Scattering (CSRS). Coherent Raman scattering is one of the means to study vibration dynamics in a time domain, and various studies thereon have been done in both aspects of experiments and theories.
This coherent Raman scattering is one of the third-order nonlinear optical response processes and generally uses two kinds of laser light (one is ω1 light and the other is ω2 light) having different angular frequencies. The laser light which firstly interacts with a molecule ω1 light and ω2 light) is also referred to as pump light and Stokes light. When the angular frequency difference between these two types of incident light is identical to the angular frequency Ω of a vibration mode of sample molecules, a large number of the sample molecules are excited in a resonant vibration mode and with the phases thereof corresponding to each other, i.e. coherently. Since the generated vibration polarization is maintained during the phase relaxation time, the molecules interact with another ω1 light as probe light during the phase relaxation time, so that coherent Raman scattered light can be taken out as a polarization wave derived from the third non-linear polarization.
More specifically, by changing delay time between the pump light and probe light, as well as Stokes light and probe light, information about phase relaxation time of molecule vibration can be obtained. In particular, as shown in FIG. 25(a), the Raman scattered light having frequency increased by +Ω is referred to as CARS. In addition, as shown in FIG. 25(b), the Raman scattered light having frequency decreased by −Ω is referred to as CSRS. In the case of CARS, since signal light is detected on the wavelength side shorter than that of excitation light, in particular, CARS is less likely to be affected by the background and the like caused by self fluorescence, whereby signal light can be detected with a good S/N ratio. Therefore, in recent years, CARS has started to be widely applied to spectroscopic microscopes and the like.
As described above, since the CARS process is nonlinear optical response process, signal light is generated only at a specific minute three-dimensional part where laser light, that is, pump light (probe light) and Stokes light are intensely focused. As a result, it is not necessary to introduce a pinhole as in a confocal microscope, whereby three-dimensional space resolution performance, which is excellent in essence, can be realized by introducing a CARS process into a scanning laser microscope. Besides, since a biological sample can be observed without being stained, the marketing prospect thereof is very promising.
2. Harmonics Process
The phenomenon attributed to the harmonics process is often observed and widely applied in the field of laser optics. For example, when laser light having a certain wavelength is made incident on an optical medium having a dielectric structure in which wavelengths are spatially in order, harmonic wave is generated from this optical medium. Specifically, a dielectric body is subjected to forced oscillation in its oscillating electric field by intense laser light and generates as a second-order wave an oscillating electric field having distortion that is not proportional to the amplitude of the oscillating electric field (a nonlinear optical effect). As a result, coherent light having a component multiplied by an integer with respect to the oscillation frequency of the incident laser light is generated. Especially, in a case where a crystal having an appropriate periodic nature is used, an extremely intense, frequency-doubled harmonic wave is generated. Therefore, it is possible to convert incident light into laser light having an arbitrary wavelength by adjusting the type of crystals and an incident angle.
This phenomenon is also applied to microscopy. For example, in a case where an observation sample has a periodic structure, when it is irradiated by coherent laser light, the sample generates a second-harmonic wave having an oscillation frequency twice as much as that of the illumination light due to its periodic structure. In other words, light having a halved wavelength occurs. By catching this second-harmonic light, a microscopic image can be obtained. Accordingly, the harmonic process is attracting attention in recent years since it allows a sample to be observed without being stained as in Raman process. The harmonic process basically utilizes a nonlinear optical effect as in Raman process.
3. Multiphoton Absorption Processes
A molecule usually has an electronic state determined by a bonding state of a valence electron of a constituent atom and has a quantum-mechanically discrete energy level. Further, an oscillation state in which bond distances between atomic nucleuses oscillate and a state such as rotation movement of the entire molecule are overlapped. In particular, when a molecule is illumination-excited from the lowest state, that is, the ground state to the next excited electronic state (the first electronically-excited state, see FIG. 24), intense fluorescence is generated from the first electronically-excited state. A typical fluorescent microscope captures this fluorescence for imaging. In this case, it is necessary to use illumination light having higher photon energy than an energy gap between the ground state and the first electronically-excited state.
On the other hand, a laser source emitting pulsed light having extremely high energy peak power is commercially available recently and thus a fluorescent microscopy (a multiphoton microscope) utilizing a multiphoton absorption process is available. In this multiphoton absorption process based on the nonlinear optical effect as in the harmonic process, when a molecule is excited from the ground state to the first electronically-excited state, the molecule is made to absorb a plurality of photons at the same time to generate fluorescence. What is characteristic of this process is that it requires photon energy of the illumination light which is much less than the above-mentioned energy gap. For example, a molecule simultaneously absorbs two photons in a two-photon process so that photon energy, which is a half of the above-mentioned energy gap, suffices.
This means that, with regard to wavelength, illumination light having wavelength twice as long as that of conventional illumination light suffices. Therefore, it is possible to normally use visible light as illumination light and, in the case of a two-photon process, use excitation light of near-infrared, whereby compact and stable semiconductor laser and fiber laser can be utilized. Further, since the responsive amount of fluorescence is proportional to square of irradiation intensity, a fluorescence responsive region is limited in a very small region in a three-dimensional space. In a case where light is focused, the responsive region in the optical axis direction becomes very small, whereby three-dimensional space resolution can be obtained, which is impossible in conventional fluorescent microscopy.